Bakanlığı, ordu #W81XWH-08-1-0587, #W81XWH-15-1-0006; Topu Devlet Üniversitesi 2010 ASPiRE grant.

<ol>
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B., Milgrom, C., Fyhrie, D., Forwood, M., Nyska, M., Finestone, A., Hoshaw, S., Saiag, E., Simkin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+measurement+of+human+tibial+strains+during+vigorous+activity.">In vivo measurement of human tibial strains during vigorous activity.</a> Bone. 18, 405-410 (1996).</li><li>Ekenman, I., Halvorsen, K., Westblad, P., Fellander-Tsai, L., Rolf, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reliability+and+validity+of+an+instrumented+staple+system+for+in+vivo+measurement+of+local+bone+deformation.+An+in+vitro+study.">The reliability and validity of an instrumented staple system for in vivo measurement of local bone deformation. An in vitro study.</a> Scand J Med Sci Sports. 8, 172-176 (1998).</li><li>Lanyon, L. E., Hampson, W. G., Goodship, A. E., Shah, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+deformation+recorded+in+vivo+from+strain+gauges+attached+to+the+human+tibial+shaft.">Bone deformation recorded in vivo from strain gauges attached to the human tibial shaft.</a> Acta Orthop Scand. 46, 256-268 (1975).</li><li>Ekenman, I., Halvorsen, K., Westblad, P., Tsai, L. F., Rolf, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+bone+deformation+at+two+predominant+sites+for+stress+fractures+of+the+tibia:+an+in+vivo+study.">Local bone deformation at two predominant sites for stress fractures of the tibia: an in vivo study.</a> Foot Ankle Int. 19, 479-484 (1998).</li><li>Milgrom, C., Finestone, A., Levi, Y., Simkin, A., Ekenman, I., Mendelson, S., Millgram, M., Nyska, M., Benjuya, N., Burr, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+high+impact+exercises+produce+higher+tibial+strains+than+running?.">Do high impact exercises produce higher tibial strains than running?.</a> Br J Sports Med. 34, 195-199 (2000).</li><li>Milgrom, C., Finestone, A., Simkin, A., Ekenman, I., Mendelson, S., Millgram, M., Nyska, M., Larsson, E., Burr, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-vivo+strain+measurements+to+evaluate+the+strengthening+potential+of+exercises+on+the+tibial+bone.">In-vivo strain measurements to evaluate the strengthening potential of exercises on the tibial bone.</a> J Bone Joint Surg Br. 82, 591-594 (2000).</li><li>Al Nazer, R., Rantalainen, T., Heinonen, A., Sievanen, H., Mikkola, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flexible+multibody+simulation+approach+in+the+analysis+of+tibial+strain+during+walking.">Flexible multibody simulation approach in the analysis of tibial strain during walking.</a> J Biomech. 41, 1036-1043 (2008).</li><li>Al Nazer, R., Klodowski, A., Rantalainen, T., Heinonen, A., Sievanen, H., Mikkola, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+full+body+musculoskeletal+model+based+on+flexible+multibody+simulation+approach+utilised+in+bone+strain+analysis+during+human+locomotion.">A full body musculoskeletal model based on flexible multibody simulation approach utilised in bone strain analysis during human locomotion.</a> Comput Method Biomec. 14, 573-579 (2011).</li><li>Johnson, M. A., Moradi, M. H., Crowe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> PID control: new identification and design methods. , 543 (2005).</li><li>Craig, R. R., Bampton, M. C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+of+substructures+for+dynamics+analysis.">Coupling of substructures for dynamics analysis.</a> American Institute of Aeronautics and Astronautics Journal. 6, 1313-1319 (1968).</li><li>Wasfy, T. M., Noor, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+strategies+for+flexible+multibody+systems.">Computational strategies for flexible multibody systems.</a> Appl Mech Rev. 56, 553-613 (2003).</li><li>Kadaba, M. P., Ramakrishnan, H. k., Wootten, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+lower+extremity+kinematics+during+level+walking.">Measurement of lower extremity kinematics during level walking.</a> J Orthop Res. 8, 383-392 (1990).</li><li>Schwartz, M. H., Rozumalski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+estimating+joint+parameters+from+motion+data.">A new method for estimating joint parameters from motion data.</a> J Biomech. 38, 107-116 (2005).</li><li>Devita, P., Skelly, W. 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Yazarlar onlar rakip hiçbir mali çıkarları var bildirin.

Bu çalışmada tibia deformasyon yüksek darbe etkinlikleri sırasında belirlemek için non-invaziv bir yöntem geliştirmekti. Tibia zorlanma nedeniyle etkisi yükleme miktarının daha iyi tibia stres kırığı anlamak için yol açacaktır. Bu çalışmada bir konuya özgü kas-iskelet modeli geliştirilmiştir ve bilgisayar simülasyonları bir laboratuvar ortamında gerçekleştirilen damla-Açılış hareketleri çoğaltmak için işletilmiştir. Tibial zorlanma damla-açılış yükseklik etkisi incelenmiştir....

Kemik stres, stres kırıkları gibi uzun süre kurtarma gerektiren ve önemli tıbbi maliyet1,2üstlenmeden şiddetli aşırı yaralanmaları yaralar. Stres kırıkları ortak atletik ve askeri nüfus her ikisi de. Arasında tüm spor yaralanmaları, stres kırıkları hesap için toplam3yüzde 10'u ile ilgili. Özellikle, sporcular yüzü daha yüksek bir yaralanma oranı % 204izlemek. Askerler de stres kırıkları yüksek orandaki deneyim. Örneğin, %6 yaralanma oranı için ABD Ordusu1 bildirildi ve % 31 yaralanma oranı İsrail Ordusu5' te bildirildi. Tüm bildirilen stres kırıkları arasında tibia stres kırığı en yaygın bir6,7,8' dir.
Spor ve fiziksel eğitimler tibia stres kırığı daha yüksek bir risk ile normalde yüksek zemin etkileri (örneğin, atlama, açılış ve kesme) ile ilişkilidir. Ayak yere kurduğunda hareket sırasında vücut için bir yere darbe kuvveti uygulanır. Bu darbe kuvveti kas-iskelet sistemi ve ayakkabı tarafından harcanmış. İskelet sistemi kolları kas Kuvvetleri zemin etkisi9emmek için uygulamak izin veren bir dizi olarak hizmet vermektedir. Bacak kasları yeterli zemin etkisi azaltmak olamaz zaman, alt-vücut kemikleri kalan gücü absorbe gerekir. Kemik yapısı bu işlemi sırasında deformasyon yaşayacaksınız. Artık darbe kuvveti tekrarlayan emilimini birikir ve stres kırıkları haline kemik microdamages içinde neden olabilir. Bugüne kadar kemik için ilgili bilgiler dış kara etkisi Kuvvetleri tepki sınırlıdır. Tibia kemik dinamik hareketleri sırasında yüksek etki güçleri tarafından tanıtılan mekanik yük nasıl yanıt vereceğini çalışma önemlidir. Tibia kemik deformasyonu yüksek darbe etkinlikleri sırasında inceleyerek tibia stres kırığı mekanizmasının daha iyi anlamak için neden olabilir.
Kemik deformasyonu vivo ölçmek için kullanılan geleneksel teknikleri Araçlı gerilim ölçerler10,11,12,13,14,15tarihinde güveniyor. Cerrahi işlemler kemik yüzeyindeki gerginlik ölçme implant için ihtiyaç vardır. İnvaziv doğası nedeniyle, in vivo çalışmalar gönüllü küçük bir örnek ile sınırlıdır. Buna ek olarak, ağırlık ölçme esneklik detektörler sadece kemik yüzeyine küçük bir bölgenin izleyebilirsiniz. Son zamanlarda, non-invaziv bir yöntem kemik zorlanma çözümlemek için bilgisayar simülasyonu kullanan tanıtılan16,17yaşındaydım. Bu metodoloji insan hareketi sırasında kemik suşu çalışmaya kas-iskelet modelleme ve bilgisayar simülasyonları birleştirme olanağını sağlar.
Bir kas-iskelet modeli bir iskelet ve iskelet kasları tarafından temsil edilir. İskelet rijit veya deforme olmayan organları olan kemik parçadan oluşur. İskelet kasları ilerici-integral-türev (PID) algoritması kullanılarak denetleyicileri olarak modellenmiştir. Üç dönemlik PID kontrol hataları tahmininde çıkış doğruluğu18geliştirmek için kullanır. Özünde, PID denetleyicileri kasları temsil eden kasların uzunluğu değişiklikler zamanla üretmek için gerekli kuvvetler geliştirerek vücut hareketleri oluşturmaya çalışın. PID denetleyicisi hata uzunluğu/saat tonundaki hareketi çoğaltılması için kuvvet değiştirmek için kullanır. Bu simülasyon işlemi tüm kas iskelet hareket ve vücut hareketleri üretmek amacıyla birlikte çalışmak için koordine etmek için uygun bir çözüm oluşturur.
İskelet kas-iskelet modeli bir veya daha fazla segmentlerinde deformasyon ölçümü izin vermek için esnek organları modellenebilir. Örneğin, tibia kemik öğeleri ve düğümleri binlerce oluşan bir sonlu sayıda öğe, bölünebilir. Esnek tibia mekanik yükleme etkisi Sonlu elemanlar (FE) analizi ile incelenebilir. FE analiz tek tek öğeleri yükleme tepki zaman içinde hesaplar. Kemik öğeleri ve düğümleri artış sayısı olarak, FE analizin hesaplama saat önemli ölçüde artacaktır.
Esnek organları deformasyon doğru değerlendirilmesi ile Hesaplamalı maliyetini azaltmak için kalıcı FE analiz geliştirilmiş ve içinde otomotiv ve Havacılık ve uzay sanayi19,20kullanılır. Bireysel FE elements yanıt zaman etki alanındaki mekanik yük için analiz yerine, bu yordamı farklı titreşim frekansları frekans etki alanındaki temel nesnenin mekanik yanıt değerlendiriyor. Bu yöntem deformasyon20doğru ölçüm sağlarken hesaplama zaman önemli bir azalma olur. Her ne kadar kalıcı FE analizi yaygın mekanik yorgunluk otomotiv ve Havacılık alanlarında eğitim için kullanılmıştır, bu yöntemin uygulama çok sınırlı olmuştur insan hareketi bilim. Al Nazer vd., insan yürüyüş sırasında tibial deformasyon incelemek için kalıcı bir FE analiz kullanılan ve sonuçları16,17teşvik bildirdi. Ancak, bu yöntem büyük ölçüde bilgisayar simülasyonları sürücü için sadece bir deney sınırlı kinematik verileri kullanarak etkilendi; Gerçek olduğunu zemin simülasyonlar yardımcı olmak için kullanılan etki güçleri. Bu yaklaşım yürüyüş gibi düşük etkisi yavaş hareketleri eğitimi için makul olabilir ama bu yüksek zemin etkisi hareketleri çalışmaya uygun bir çözüm değil. Böylece, dinamik yüksek darbe faaliyetleri sırasında alt vücut kemik reaksiyonları incelemek için daha önce bildirilen yöntemiyle ilişkili sınırlamaları gidermek için yenilikçi bir yaklaşım geliştirmek esastır. Özellikle, bir yöntem kullanmak doğru deneysel kinematik veri ve gerçek kara etkisi Kuvvetleri geliştirilen gerekir. Bu nedenle, bu çalışmanın amacı multibody dinamik simülasyonlar tibial zorlanma yüksek darbe faaliyetleri sırasında incelemek için kalıcı FE analizi ile gerçekleştirmek için bir konuya özgü kas-iskelet modeli geliştirmekti. Damla-iniş tarafından farklı yükseklikte temsil bir dinamik yüksek darbe hareketi yöntemini sınamanızı sağlamak üzere seçildi.

<table border="0" cellpadding="0" cellspacing="0" width="575">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:143px;">CT Scanner</td>
			<td style="width:123px;">GE Medical System</td>
			<td style="width:107px;">N/A</td>
			<td style="width:203px;">Light Speed VCT. For performing tibia CT scan.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Motion Capture System</td>
			<td>Vicon Inc</td>
			<td>N/A</td>
			<td>Vicon FX40 high speed cameras. For performing 3D motion capture.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Force plates</td>
			<td>AMTI Inc</td>
			<td>N/A</td>
			<td>Collecting 3D ground reaction forces</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vicon Nexus</td>
			<td>Vicon Inc</td>
			<td>N/A</td>
			<td>Motion capture software program. For processing visual marker trajectory data.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Visual 3D</td>
			<td>C-Motion Inc</td>
			<td>N/A</td>
			<td>Biomechanics analysis software. For computing 3D kinematics and kinetics of human movements.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MATLAB</td>
			<td>Mathworks Inc</td>
			<td>N/A</td>
			<td>Computer programming software. For performing raw data filtering, data conversion, and data processing.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ADAMS 2012</td>
			<td>MSC Software Inc</td>
			<td>N/A</td>
			<td>Multibody dynamic computer simulation program.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LifeMOD</td>
			<td>Lifemodeler Inc</td>
			<td>N/A</td>
			<td>A software Plug-in in ADAMS. For building human body musculo-skeletal models.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MIMICS 13</td>
			<td>Materialise Inc</td>
			<td>N/A</td>
			<td>Image processing program. A 3D modeling tool to process imaging data. For creating 3D tibia model from CT scans.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MARC 2012</td>
			<td>MSC Software Inc</td>
			<td>N/A</td>
			<td>Finite element analysis software. For performing volumn meshing, generating tibia FE model, and running modal FE analysis.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SPSS 19</td>
			<td>IBM Inc</td>
			<td>N/A</td>
			<td>Statistical analysis software.</td>
		</tr>
	</tbody>
</table>

subject-specific musculoskeletal model for studying bone strain during dynamic motion

Kemik stres kırıklarının spor ve askeri eğitimleri yaygındır. Tekrarlayan büyük kara etkisi Kuvvetleri eğitim sırasında neden olabilir. Kemik stres yaralanma mekanizmaları daha iyi anlamak için alt-vücut kemik deformasyon üzerinde yüksek zemin etkisi etkisini zorlar belirlemek için önemlidir. Geleneksel ağırlık ölçme esneklik detektörler ölçüm içinde vivo tibia deformasyon çalışma kullanılmaya başlanmıştır. Bu yöntem invasiveness yordam, kaç insan denekler ve küçük kemik yüzey alanları sınırlı zorlanma verileri katılımı da dahil olmak üzere sınırlamalar ile ilişkilidir. Çalışmada tibia kemik baskı koşulları yükleme yüksek etki altında çalışmaya yeni bir yaklaşımın tanıtmak niyetinde. Bir konuya özgü kas-iskelet modeli sağlıklı erkek (19 yıl, 80 kg, 1800 mm) temsil etmek için oluşturuldu. Esnek Sonlu elemanlar tibia modeli bir bilgisayarlı tomografi (CT) inceden inceye gözden geçirmek konunun doğru Tibia üzerinde tabanlı oluşturuldu. Laboratuvar hareket yakalama damla-iniş kinematik ve zemin tepki kuvvetlerinin farklı yükseklikte (26, 39, 52 cm) elde etmek için gerçekleştirildi. Esnek tibia kalıcı bir analizi ile kombine multibody dinamik bilgisayar simülasyonları tibia zorlanma damla-iniş sırasında ölçmek için yapıldı. Hesaplanan tibia zorlanma veri önceki in vivo çalışmalar ile iyi anlaşma vardı. Non-invaziv bu yaklaşım daha iyi tibia stres kırıkları yaralanma mekanizmasının anlaşılması için yol açacak büyük bir kohort için yüksek darbe faaliyetleri sırasında tibia kemik suşu çalışmaya uygulanabilir belirgindir.

Çalışma için gönüllü oldum bir sağlıklı kafkas erkek (19 yaş, yükseklik 1.800 mm, kitle 80 kg). Veri toplama önce konu incelendi ve çalışmaya katılan önce üniversite kurumsal inceleme Kurulu tarafından onaylanan izin formu imzaladı. Helsinki Bildirgesi altında yapılan. Deneme temel alınarak aşağıdaki protokol gerçekleştirildi.
İleri dinamik simülasyon doğruluğunu denetlemek için alt-vücut simü...

Watch this Scientific Journal Video about Subject-specific Musculoskeletal Model for Studying Bone Strain During Dynamic Motion at JoVE.com

Dinamik Hareket Sırasında Kemik Gerilmelerinin İncelenmesi için Konuya Özel Kas-İskelet Modeli | Biyomühendislik | Video

Subject-specific Musculoskeletal Model for Studying Bone Strain During Dynamic Motion

İniş sırasında alt vücut kemikleri büyük mekanik yükler deneyim ve deforme. Etkileri ile ilişkili kemik stres yaralanma mekanizmaları daha iyi anlamak için kemik deformasyonu ölçmek için önemlidir. Konuya özgü kas-iskelet modelleme ve Sonlu elemanlar analizi entegre yeni bir yaklaşımın tibial zorlanma sırasında dinamik hareketleri ölçmek için kullanılır.

Konuya özgü kas-iskelet modeli kemik zorlanma sırasında dinamik hareket eğitimi

Bone stress injuries are common in sports and military trainings. Repetitive large ground impact forces during training could be the cause. It is ...

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9.6K Görüntüleme.  Ball State University. İniş sırasında alt vücut kemikleri büyük mekanik yükler deneyim ve deforme. Etkileri ile ilişkili kemik stres yaralanma mekanizmaları daha iyi anlamak için kemik deformasyonu ölçmek için önemlidir. Konuya özgü kas-iskelet modelleme ve Sonlu elemanlar analizi entegre yeni bir yaklaşımın tibial zorlanma sırasında dinamik hareketleri ölçmek için kullanılır.

Video: Subject-specific Musculoskeletal Model for Studying Bone Strain During Dynamic Motion

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

Mekanik Dinamik Hücre Kültürü için Microfabricated platformlar

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Biyomühendislik

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

Protein Katlama Öğrenmenin mikroakışkan Mikserler

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Dinamik Etkileşimler 3D Yapısal Analiz için karşılıklı Mikroskopi

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Home-Based Monitor for Gait and Activity Analysis

Current outcomes in neuromuscular disorder clinical trials include motor function scales, timed tests, and strength measures performed by trained clinical evaluators. These measures are slightly subjective and are performed during a visit to a clinic or hospital and constitute therefore a point assessment. Point assessments can be influenced by daily patient condition or factors such as fatigue, motivation, and intercurrent illness. To enable home-based monitoring of gait and activity, a wearable magneto-inertial sensor (WMIS) has been developed. This device is a movement monitor composed of two very light watch-like sensors and a docking station. Each sensor contains a tri-axial accelerometer, gyroscope, magnetometer, and a barometer that record linear acceleration, angular velocity, the magnetic field of the movement in all directions, and barometric altitude, respectively. The sensors can be worn on the wrist, ankle, or wheelchair to record the subject&#8217;s movements during the day. The docking station enables data uploading and recharging of sensor batteries during the night. Data are analyzed using proprietary algorithms to compute parameters representative of the type and intensity of the performed movement. This WMIS can record a set of digital biomarkers, including cumulative variables, such as total number of meters walked, and descriptive gait variables, such as the percentage of the most rapid or longest stride that represents the top performance of patient over a predefined period of time.

This innovative device uses magneto-inertial sensors to permit gait and activity analysis in uncontrolled environments. Currently in the qualification process as an outcome measure in the European Medical Agency, one of the applications will be to serve as a clinical endpoint in clinical trials in neuromuscular diseases.

Yürüyüş ve aktivite analizi için ev tabanlı monitör

Current outcomes in neuromuscular disorder clinical trials include motor function scales, timed tests, and strength measures performed by trained clinical ...

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows assessment of bone substitution materials for oral and craniofacial bone regeneration in terms of bone growth and neovascularization support. Once animals are anesthetized and ventilated (endotracheal intubation), four cylinders made of polyether ether ketone (PEEK) are screwed onto the skull, on both sides of the median and coronal sutures. Five intramedullary holes are drilled within the bone area delimited by each cylinder, allowing influx of bone marrow cells. The material samples are placed into the cylinders which are then closed. Finally, the surgical site is sutured, and animals are awaken. Bone growth may be assessed on live animals by using microtomography. Once animals are euthanized, bone growth and neovascularization may be evaluated by using microtomography, immune-histology and immunofluorescence. As the evaluation of a material requires maximum standardization and calibration, the calvarial model appears ideal. Access is very easy, calibration and standardization are facilitated by the use of defined cylinders and four samples may be assessed simultaneously. Furthermore, live tomography may be used and ultimately a large decrease in animals to be euthanized may be anticipated.

Here we present a surgical protocol in rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. By using PEEK cylinders fixed onto rabbit skulls, osteoconduction, osteoinduction, osteogenesis and vasculogenesis induced by the materials may be evaluated either on live or euthanized animals.

Kemik İkmal Malzemelerinde Kemik Büyümesi ve Neaskülarizasyonunun Değerlendirilmesi İçin Tavşanda Kemik Büyütmenin Kalvarial Modeli

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

Bir İnsan 3D Ekstrasellüler Matrix-Adiposit Kültür Modeli Matrix-Hücre Metabolik Crosstalk Çalışma için

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

Canlı Mikroskopi Görüntüleme Altında 3D Hidrojellerin Kontrollü Suşu

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

Measuring 3D In-vivo Shoulder Kinematics using Biplanar Videoradiography

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, multiple ligaments, and approximately 20 muscles. Unfortunately, shoulder pathologies (e.g., rotator cuff tears, joint dislocations, arthritis) are common, resulting in substantial pain, disability, and decreased quality of life. The specific etiology for many of these pathologic conditions is not fully understood, but it is generally accepted that shoulder pathology is often associated with altered joint motion. Unfortunately, measuring shoulder motion with the necessary level of accuracy to investigate motion-based hypotheses is not trivial. However, radiographic-based motion measurement techniques have provided the advancement necessary to investigate motion-based hypotheses and provide a mechanistic understanding of shoulder function. Thus, the purpose of this article is to describe the approaches for measuring shoulder motion using a custom biplanar videoradiography system. The specific objectives of this article are to describe the protocols to acquire biplanar videoradiographic images of the shoulder complex, acquire CT scans, develop 3D bone models, locate anatomical landmarks, track the position and orientation of the humerus, scapula, and torso from the biplanar radiographic images, and calculate the kinematic outcome measures. In addition, the article will describe special considerations unique to the shoulder when measuring joint kinematics using this approach.

Biplane videoradiography can quantify shoulder kinematics with a high degree of accuracy. The protocol described herein was specifically designed to track the scapula, humerus, and the ribs during planar humeral elevation, and outlines the procedures for data collection, processing, and analysis. Unique considerations for data collection are also described.

Biplanar Videoradyografi kullanarak 3D In-vivo Omuz Kinematik ölçümü

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, ...

Cantilever Bending of Murine Femoral Necks

Fractures in the femoral neck are a common occurrence in individuals with osteoporosis. Many mouse models have been developed to assess disease states and therapies, with biomechanical testing as a primary outcome measure. However, traditional biomechanical testing focuses on torsion or bending tests applied to the midshaft of the long bones. This is not typically the site of high-risk fractures in osteoporotic individuals. Therefore, a biomechanical testing protocol was developed that tests the femoral necks of murine femurs in cantilever bending loading to replicate better the types of fractures experienced by osteoporosis patients. Since the biomechanical outcomes are highly dependent on the flexural loading direction relative to the femoral neck, 3D printed guides were created to maintain a femoral shaft at an angle of 20&#176; relative to the loading direction. The new protocol streamlined the testing by reducing variability in alignment (21.6&#176; &plusmn; 1.5&#176;, COV = 7.1%, n = 20) and improved reproducibility in the measured biomechanical outcomes (average COV = 26.7%). The new approach using the 3D printed guides for reliable specimen alignment improves rigor and reproducibility by reducing the measurement errors due to specimen misalignment, which should minimize sample sizes in mouse studies of osteoporosis.

The present protocol describes the development of a reproducible testing platform for murine femoral necks in a cantilever bending set-up. Custom 3D printed guides were used to consistently and rigidly fix the femurs in optimal alignment.

Murine Femoral Boyunların Kantilever Bükülmesi

Fractures in the femoral neck are a common occurrence in individuals with osteoporosis. Many mouse models have been developed to assess disease states and ...

A GMP-Compliant Procedure for the Generation of Gene-Modified T cells

The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor (CAR)-T cells. These modified cells have shown remarkable clinical responses in patients with hematologic malignancies. However, the high cost of producing these therapies and conducting extensive quality control assessments has limited their accessibility to a broader range of patients. To address this issue, many academic institutions are exploring the feasibility of in-house manufacturing of genetically modified cells, while adhering to guidelines set by national and international regulatory agencies.
Manufacturing genetically modified T cell products on a large scale presents several challenges, particularly in terms of the institution's production capabilities and the need to meet infusion quantity requirements. One major challenge involves producing large-scale viral vectors under Good Manufacturing Practice (GMP) guidelines, which is often outsourced to external companies. Additionally, simplifying the T cell transduction process can help minimize variability between production batches, reduce costs, and facilitate personnel training. In this study, we outline a streamlined process for lentiviral transduction of primary human T cells with a fluorescent marker as the gene of interest. The entire process adheres to GMP-compliant standards and is implemented within our academic institution.

This protocol outlines the process of transducing primary human T cells with a gene of interest, ensuring compatibility with Good Manufacturing Practice (GMP) standards.

Geni Değiştirilmiş T hücrelerinin Üretimi için GMP Uyumlu Bir Prosedür

The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor ...